Highly sensitive detection systems have become important analytical tools in medical diagnostics and biological research, as illustrated by the use of fluorescence immunochemistry, flow cytometry, enzyme-linked immunocytochemistry, and in situ DNA or RNA hybridization. In some areas, nevertheless, there is a substantial interest in developing new cost-effective techniques which extend presently available detection limits. For example, some patients with early-stage cancer have small number of metastatic tumor cells in their bone marrow which escape detection by routine procedures such as bone scan, biochemical analysis, and cytological examinations. The most frequently used methodology for detecting these rare tumor cells, usually referred as occult micrometastases, is immunocytochemistry (Premi, T., and Battifora, H. Human Pathol. 18, 728-734, 1987, hereby incorporated as a reference). At present, however, immunocytochemistry cannot be used on a routine basis because it is extremely labor-intensive. For example, a histopathology-trained technician necessitates about four hours of microscopic scanning to analyze a specimen (and appropriate controls) containing less than 10 malignant cells per million bone marrow cells.
Automated screening techniques, such as flow cytometry and computerized image analysis, not only require extensive capital investments but have been found to be less sensitive than conventional immunocytochemistry. It is clear, therefore, that in this particular field there is a need for simple and cost-effective techniques capable of detecting malignant cells present at low frequencies in clinical specimens of blood or bone marrow. Other clinical fields may also benefit from such techniques because, for example, they could provide sensitive means to: 1) detect early relapse in cancer patients; 2) test effectiveness of adjuvant treatments throughout therapy of patients with metastastic disease; 3) monitor the presence of tumor cells in blood or bone marrow used for autologous transplantation to prevent infusion of tumor cells into patients; 4) track circulating genetically engineered cells in patients.
Similarly, simple, more sensitive techniques could find clinical and investigational applications for lowering the detection levels of biologically important macromolecules which are presently analyzed using enzyme-linked or radioactive probes. For example, amplification-visualization techniques currently used for DNA-probe detection include enzyme-catalyzed reactions yielding chemiluminescent products, fluorescent products, or colored insoluble precipitates. Typically, in these techniques, a labeled nucleic acid probe is annealed to a complementary DNA or RNA target sequence which is either in solution or immobilized on an inert support. The binding of the labeled probe (usually an oligonucleotide which either contains a radioactive element or is attached to an enzyme via conventional ligand-binding protein technology) reports the presence or absence of a the target sequence in the reaction mixture. Examples of clinical applications using DNA probe amplification-visualization are tests for viruses, oncogenes, or multiple resistance genes using enzyme-labeled DNA probes.